A spectrofluorometric method is commonly used as a detection method for chromatograph systems. Although the spectrofluorometric method is only available for the detection of fluorescent substances, this technique is broadly used, because it is capable of an analysis with dramatically high sensitivity as compared to an optical absorption method, which utilizes absorption of light by a substance. FIG. 1 is a schematic configuration diagram of a spectrofluorometric detector for performing a spectrofluorometric method.
In this spectrofluorometric detector 1, light generated by a light source (e.g. Xenon lamp) having a broad continuous spectrum ranging from the ultraviolet region through the near infrared region is introduced into an excitation-light dispersing device 11, which turns the light into a monochromatic light of a specific excitation-light wavelength by a diffraction grating 11a driven by a motor. This monochromatic light is cast as excitation light into a sample cell 12 containing a sample solution 13. Upon being irradiated with the excitation light, the sample solution 13 emits a faint fluorescence, which is introduced into a fluorescence dispersing device 14. In this device, only a fluorescence of a specific wavelength is extracted by a diffraction grating 14a and sent to a photomultiplier tube 15. This tube 15 produces a current signal corresponding to the intensity of the incident light. The current signal is converted into a voltage signal by a current-to-voltage (I/V) converter 16 and further into a digital value by an analogue-to-digital (A/D) converter 17, to be ultimately sent to a data processor 18 as detection data. By processing and analyzing the detection data, the data processor 18 calculates a quantitative value of a specific component in the sample solution 13.
Normally, in a spectrofluorometric method, a three-dimensional fluorescent spectrum having the three axes of excitation-light wavelength, fluorescence wavelength and fluorescence intensity is obtained by repeating the measurement in which either the excitation-light wavelength or fluorescence wavelength is sequentially changed while the other wavelength is fixed.
This method requires a considerable length of time to search for peaks since the excitation-light wavelength and the fluorescence wavelength must be independently and sequentially changed to obtain a fluorescent spectrum. To address this problem, a method for efficiently searching for peaks has been proposed in Patent Document 1. To improve the peak-searching efficiency, this method utilizes the characteristic fact that a large number of peaks on a fluorescent spectrum obtained by sequentially changing the excitation-light wavelength and the fluorescence wavelength have their fluorescence wavelengths being longer than their excitation-light wavelengths by 20 nm to 140 nm. According to this method, a fluorescent spectrum is obtained by simultaneously changing the excitation-light wavelength and the fluorescence wavelength while controlling these wavelengths so that the fluorescence wavelength is always longer than the excitation-light wavelength by 20 to 140 nm.
In this method, a rough yet efficient search of a peak is initially performed by simultaneously changing the excitation-light wavelength and the fluorescence wavelength. After a peak is found, the excitation-light wavelength and the fluorescence wavelength are independently changed to obtain detailed information. Accordingly, the measurement requires less time than in the case of the conventional spectrofluorometric method in which each of the excitation-light and fluorescence wavelengths is independently changed over the entire range of the measurement wavelength.
To measure a fluorescent spectrum by this method, a preliminary measurement or the like is previously performed to collect, for each of the excitation-light and fluorescence dispersing devices, information relating to the number of pulses to be sent to the pulse motor for driving the light-dispersing element and the change in the wavelength of the monochromatic light produced by the light-dispersing element. Using this information, it is possible to determine the numbers of pulses to be respectively sent to the excitation-light pulse motor and the fluorescence pulse motor so as to concurrently change the excitation-light wavelength and the fluorescence wavelength under given spectrofluorometric measurement conditions (the measurement-beginning wavelength, the measurement-ending wavelength, and the wavelength-changing interval).